live yeast dietary supplementation acts upon intestinal morpho

Animal Feed Science and Technology129 (2006) 224–236

Live yeast dietary supplementation acts uponintestinal morpho-functional aspects and

growth in weanling piglets

V. Bontempo ∗, A. Di Giancamillo, G. Savoini,V. Dell’Orto, C. Domeneghini

Department of Veterinary Sciences and Technologies for Food Safety,University of Milan, Via Celoria 10, 20133 Milan, Italy

Received 10 September 2004; received in revised form 6 December 2005; accepted 29 December 2005

Abstract

Three hundred and fifty-two piglets were at weaning assigned to two dietary experimental groups,Ctr (control) or Y (yeast-supplemented). The yeast supplement (2 g/kg of diet) provided 2×106 CFU/gof feed. Piglet growth was monitored from weaning to 4 weeks after weaning. On day 28 post-weaning,20 female piglets (10 per group) were slaughtered and the distal ileum was sampled from each animal,and examined with microanatomical methods (histology, histochemistry, immunohistochemistry, andhistometry). Villus and crypt measurements were taken, mucin profile assessed with histochemistry,and mucosal macrophages and proliferating epithelial cells determined after immunolocalization.Yeast supplementation to piglets was associated with a greater live weight (P<0.001) and a greater post-weaning daily gain (P<0.001) in comparison with control animals. The intestinal adherent mucouslayer was thicker in control than supplemented piglets (P<0.001). Proliferating epithelial cell countswere greater in supplemented than control piglets (P=0.045). Mucosal macrophages were more numer-ous in supplemented than control piglets (P=0.007). These findings indicate that live yeast dietarysupplementation to piglets is able to improve nursery growth performance. Effects on intestinal mucosasuggest that supplementation is able to promote a “healthy” intestine, encouraging an early restora-

∗ Corresponding author. Tel.: +39 02 58357900; fax: +39 02 58357898.E-mail address: [email protected] (V. Bontempo).

0377-8401/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.anifeedsci.2005.12.015

V. Bontempo et al. / Animal Feed Science and Technology 129 (2006) 224–236 225

tion of the intestinal mucosal thinning that often occurs at weaning, and possibly improving localresistance to infection.© 2006 Elsevier B.V. All rights reserved.

Keywords: Live yeast; Piglet; Ileum; Mucine profile; Immunohistochemistry; Histometry

1. Introduction

Piglets undergo a stress-related growth check at weaning, often associated with anorexiaand under-nutrition, with predisposition to diarrhoea and intestinal infections. Several alter-natives to antibiotics have been proposed for managing piglet gut health at this crucial period,including the administration of probiotics. Probiotics are preparations of non-pathogenicmicroorganisms, prepared for animal and human use, that may have beneficial effects onthe digestive ecosystem and confer resistance to infection (Fuller, 1992).

Live yeast preparations are probiotics rich in enzymes, vitamins, nutrients and co-factors.Yeasts are highly resistant to inactivation during their passage along the alimentary canal,and in humans may colonize the gut to help in restoring disturbed gastrointestinal microecol-ogy (Girola and Ventura, 1995; Bleichner et al., 1997). They have been reported to producea variety of beneficial production responses in animals, including piglets (Jurgens et al.,1997; Maloney et al., 1998; Mathew et al., 1998; Van Heugten et al., 2003). However, otherstudies have reported the absence of benefits from live yeast supplementation (Kornegay etal., 1995).

Saccharomyces cerevisiae ssp. boulardii is a non-pathogenic yeast that, when orallygiven to laboratory mammals, has been reported to stimulate gut-associated-lymphoid tis-sue (GALT) resulting in enhanced secretion of specific IgA (Buts et al., 1990), to inhibitbinding of bacterial toxins to enterocyte receptors (Pothoulakis et al., 1993), and to enhanceendoluminal production of polyamines thereby reducing intestinal permeability (Kollmanet al., 2001; Costalos et al., 2003; Buts et al., 1994).

The aim of the present study was to investigate the effects of dietary supplementationwith live S. boulardii on morpho-functional aspects of piglet intestine, and on aspects ofpiglet performance during the first month after weaning.

2. Materials and methods

A total of 352 piglets from weaning (25 day of age) until a month after weaning were usedunder farm conditions. Piglets were weaned by moving them to environmentally controlledpens. There were 16 pens with 20–25 piglets per pen; males and females were separated.Each pen was 0.9 m×2 m, had a slatted floor, and was equipped with water nipple andfour-hole self-feeder. The piglets were allowed an ad libitum access to feed and water.

2.1. Diet

Piglets were randomly allotted to control (Ctr) or yeast dietary supplementation (Y) with8 replicates per treatment. The yeast supplement used was a concentrate of live S. cerevisiae

226 V. Bontempo et al. / Animal Feed Science and Technology 129 (2006) 224–236

Table 1Composition of piglet diet, as-fed basis

Ingredients g/kg

IngredientsMaize 356Barley 50Cereals, steam-rolleda 85Milk powder, spray-dried 127Whey powder, spray-dried 103Full fat whey (50% fat) 15Fish meal, menhaden 75Soybean meal (48% CP) 130Maize oil 25Coconut oil 5Dextrose 10Limestone, ground 7Dicalcium phosphate 5Salt 1Vitamin–mineral premixb 2.6Copper sulfate 0.8Zinc oxide 0.3l-Lysine HCl 1.5dl-Methionine 0.6Tryptophan 0.2Yeastc +/−

Calculated compositionME (MJ/kg) 13.8CP (g/kg) 210.2Lysine (g/kg) 14.1Ca (g/kg) 9.1P (g/kg) 7.5

a Mixed cereals: 250 g maize, barley, oats and wheat/kg.b Supplying a minimum per kilogram complete diet of: 12,500 IU Vitamin A; 1250 IU Vitamin D; 125 IU Vitamin

E; 90 �g Vitamin B12; 9 mg riboflavin; 45 mg pantothenic acid; 35 mg niacin; 4.5 mg folic acid; 0.25 mg biotin;130 mg Fe; 170 mg Zn; 15 mg Cu; 30 mg Mn; 0.60 mg I; and 0.28 mg Se.

c Live yeast (Saccharomyces cerevisiae ssp. boulardii, Levucell SB, CNCM I-1079; Lallemand, France) wasadded to treated diets at 2 g/kg of feed at the expense of maize steam rolled or corn alone (calculated as 2×106 CFU/gof feed).

ssp. boulardii (Levucell SB-CNCM I-1079, Lallemand, France). Live yeast content wasover 2×106 CFU/g of feed. All piglets received a starter diet that was either control (Ctr,no added yeast) or contained 2 g/kg added live yeast (Y, 2×106 CFU/g of feed). Diets werefortified to meet or exceed NRC (1998) requirements for all nutrients (Table 1). Antibioticsas growth-promoting agents were absent. Total zinc and copper were present in the diet at270 and 30 ppm, respectively. Live weight, feed intake, and feed efficiency were recordedfor the 28-day post-weaning study.

All animals were treated in accordance with European Community guidelines approvedby the Italian Ministry of Health.


2.2. Microscopic anatomy of piglet ileum

At day 28 post-weaning, 10 female piglets per group (total number of animals = 20)were slaughtered. Females were chosen for the intestinal microscopic study because ofthe homogeneous weight within the experimental groups. The entire intestinal tracts wereremoved, and the distal ileum (2 cm prior to the opening into the caecum) was collectedfrom each animal (total number of samples = 20) and promptly fixed in 4% paraformalde-hyde in 0.01 M phosphate buffered saline (PBS) pH 7.4 for 24 h at 4 ◦C. The specimenswere then dehydrated in graded alcohols, cleared with xylene and embedded in paraffin.After dewaxing and re-hydration, serial microtome sections (4 �m thick) were stained withhematoxylin and eosin and examined to assess microanatomical structure and determinevillus height (V) (10 villi measured per section), crypt depth (C) (10 crypts measured persection), and the villus height to crypt depth ratio (V:C ratio).

The ileum mucin profile was determined by staining some sections (a) with the Alcianblue 8GX, pH 2.5-periodic acid Schiff combination (AB-PAS), which reveals neutral (PAS-reactive) and acid (AB-reactive) glycoconjugates, and (b) with the high iron diamine-Alcianblue 8GX, pH 2.5 combination (HID-AB), which demonstrates sulphated and sialylatedglycoconjugates respectively. The thickness of the adherent mucous layer, determined asthe distance from the outermost surface of it to the luminal surface of the epithelial lining,was measured at 10 randomly selected points in each AB-PAS-stained section (Matsuo etal., 1997).

Other ileum sections were processed to visualize mucosal cells, which were in the Sphase of the cell cycle (proliferating cells), by immunostaining with a monoclonal anti-serum against proliferating cell nuclear antigen (PCNA) (PC10 clone, Sigma, Italy), andthe subsequent revelation of the immunoreactive sites with the peroxidase-antiperoxidase(PAP) method. Briefly, following dewaxing and re-hydration, the sections were immersedin a freshly prepared solution of 3% H2O2 in distilled water for 10 min to block endoge-nous peroxidase activity. For the antigen retrieval, slides with sections were heated in amicrowave at 700 W twice (5 min each) in citrate buffer 0.01 M, adjusted to pH 6.0 withNaOH 2N (Foley et al., 1991; Greenwell et al., 1991; Cattoretti et al., 1993; Shi et al., 1995).After cooling at room temperature (15 min), the sections were rinsed in Tris-buffered saline(TBS) pH 7.5 and incubated with normal swine serum (Dako, Italy) at 1:5 dilution in TBScontaining 1% bovine serum albumin (BSA) for 20 min. The PCNA-antiserum was applied(dilution 1:3000 in TBS plus 1% BSA) for 45 min at room temperature in a humid chamber.Sections were then incubated with rabbit anti-mouse IgG (Dako), followed by incubationwith mouse PAP complex (Dako). Immunoreactivity was visualized using a freshly preparedsolution of 3,3′-diaminobenzidine tetrahydrochloride (Sigma). Sections were briefly coun-terstained with Mayer’s hematoxylin, dehydrated and permanently mounted with Eukitt(Bio Optica, Italy). The proliferative index was determined by counting epithelial cells withPCNA-positive nuclei in 10 well-oriented villi/crypts for each ileum section (Burrin et al.,2000a).

Other ileum sections were processed immunohistochemically to identify mucosalmacrophages using an anti-human macrophage monoclonal antiserum (LN-5 clone, Sigma)diluted 1:400 in TBS. The steps were as for PCNA immunostaining, except that antigenretrieval was not necessary. The macrophage index was determined in GALT. For each sec-


Table 2Least mean square of piglets growth performance (mean ± S.E.)

Ctr (n = 176) Y (n = 176) P

Piglets weight (kg)At weaning 7.9 ± 0.11 7.5 ± 0.11 0.01930-day post-weaning 19.4 ± 0.15 20.4 ± 0.14 <0.001Average daily gain (g) 432 ± 0.01 474 ± 0.01 <0.001Daily feed intake (g) 691 ± 0.13 670 ± 0.12 0.110

Feed:gain 1.6 ± 0.17 1.4 ± 0.15 0.180

tion, the number of immunopositive mucosal cells was counted in 10 fields (representing atissue area of about 0.015 mm2) (Sozmen et al., 1996).

The specificity of immunostaining was verified for both antisera by incubating otherileum sections with normal mouse serum (Dako) instead of the primary antisera: this alwaysgave negative results. As positive controls, alimentary canal samples from cow and dog weretested. In all cases the expected positive reactions were observed. Slides from all groupswere stained together in a single batch.

For all the observations and measurements, an Olympus BX51 microscope equippedwith DP software for the computed image analysis (Olympus, Italy) was used.

Observations and counting were performed by an observer blind to the control versusyeast-supplemented status of the sections.

2.3. Statistical analysis

ANOVA (SAS Inst., 1999) was used to analyze differences in piglet weight and averagedaily gain, including dietary supplementation, and pen within supplementation as fixedeffects, and weight at weaning as covariate. Following which, piglets’ diet and pen withindiet were analyzed by orthogonal contrast.

Histometric data (villus height, crypt depth, V:C ratio, mucous layer thickness) wereanalyzed by ANOVA using the mixed procedure of the SAS package (1999). The modelincluded the treatment as fixed effect and piglet as random effect. The total number ofmucosal cells with PCNA-positive nuclei were added as a linear regression in the model.The total number of mucosal cells with anti-macrophage-positivity was also added as alinear regression in the model. The data are presented as least squared mean ± S.E.M. Dif-ferences between least squared means were analyzed by orthogonal contrast and consideredsignificant at P<0.05.

3. Results

3.1. Effects of yeast supplementation on performance

The effects of yeast supplementation on piglet growth are shown in Table 2. Controlpiglets were heavier (P<0.05) than treated piglets at weaning, but the latter ones weresignificantly heavier at 30 days post-weaning (P<0.01). Piglets fed yeast had a significantly


Table 3Villus height (V), crypt depth (C), V:C ratio; mucin profile; mitotic cell counts; mucosal macrophage counts inpiglets ileum (mean ± pooled S.E.)

Ctr (n = 10) Y (n = 10) P

Villous height (V) (�m) 195 ± 3 243 ± 3 0.001Crypts depth (C) (�m) 130 ± 2 177 ± 2 0.001V:C ratio 1.5 ± 0.01 1.4 ± 0.01 0.088Adherent mucous gel thickness (�m) 2.9 ± 0.09 1.7 ± 0.08 <0.001Mitotic cells % of total 42 ± 2 49 ± 2 0.045Macrophage 4.0 ± 0.06 4.9 ± 0.08 0.007

greater average daily gain from weaning throughout 30 days post-weaning (P<0.01) thannon-supplemented piglets.

Piglets fed yeast also grew more efficiently (better feed:gain ratio) than those fed basaldiet from weaning through 30 days post-weaning, but the difference was not significant(Table 2).

3.2. Microscopic anatomy of piglet ileum

Features suggesting histopathological aspects in the ileum of either control or treatedpiglets were never observed. The intestinal mucosa was regularly organized in intestinalvilli and crypts in both control and yeast-supplemented piglets. The presence of GALT wasusual for this species in both its prominence and localization (Peyer’s patches).

Histometric analysis showed that villus height (P<0.01) and crypt depth (P<0.01) weresignificantly greater in yeast-fed piglets than those not given yeast. Consequently, V:C ratiowas lower (P=0.088) in yeast-fed piglets than those not given yeast (Table 3).

Fig. 1. Control piglet ileum. PCNA immunostaining, scale bar: 20 �m. Positive nuclei are present in enterocytesof intestinal crypts (arrows).


Fig. 2. Yeast-supplemented piglet ileum. PCNA immunostaining, scale bar: 20 �m. Numerous strongly immunore-active nuclei are present in enterocytes of intestinal crypts (arrows).

AB-PAS sequential staining showed that intestinal goblet cells contained both neutral andacid glycoconjugates. Acid glycoconjugates were predominant in supplemented (Y) piglets,particularly in crypts. HID-AB staining showed that, in all cases, goblet cells containingsulphated glycoconjugates predominated in villi, and goblet cells containing sialylated gly-coconjugates occurred mainly at the bases of crypts.

In AB-PAS-stained sections, the adherent mucous gel was significantly thicker in controlpiglets than supplemented animals (P<0.01).

Fig. 3. Control piglet ileum. Macrophage immunostaining, scale bar: 20 �m. Immunopositive cells are present inthe tunica propria (arrows).


Fig. 4. Yeast-supplemented piglet ileum. Macrophage immunostaining, scale bar: 20 �m. Immunopositive cellsare present in diffuse lymphatic tissue (arrows).

PCNA-immunoreactive nuclei were numerous in enterocytes of ileum samples from allgroups (Figs. 1 and 2). Epithelial cells with such immunoreactive nuclei (proliferating cells)were significantly (P<0.05) more numerous in supplemented than control piglets.

Immunostaining for macrophages picked out numerous rounded immunoreactive cells inthe GALT of all animals (Figs. 3 and 4). However, macrophages were significantly (P<0.01)more numerous in treated than non-treated piglets.

4. Discussion

The aim of the experiment was to determine the effects of dietary supplementation withlive yeast (S. cerevisiae ssp. boulardii) on piglet growth and gut structure. It was particularlyconcerned with weanling piglets since these animals undergo a stress-related growth checkat this time, and any reduction in gut tissue mass, linked for example to a gastrointestinaldisorder (Burrin et al., 2000b), can result in marked morbidity and significantly reducedgrowth performance (Pluske et al., 1997).

During post-weaning, piglets fed yeast had significantly greater average daily gain thancontrol piglets, and this may be reasonably attributed to the yeast supplementation. Similarimprovements in piglet average daily gain as a result of yeast supplementation have beenreported by some studies (Jurgens et al., 1997; Maloney et al., 1998; Mathew et al., 1998),while others have found that yeast supplementation to weanling piglets had no effect onaverage daily gain (Kornegay et al., 1995; Jurgens et al., 1997). These discrepant findingsmay be due to differences in sanitary conditions of animals, composition of diet, and quantityand type of live yeast added to diet. Van Heugten et al. (2003) founded that live yeastsupplementation had positive effects on nursery piglet performance, but only when dietscontained growth-promoting anti-microbial substances.


Histometric analysis showed that villus height and crypt depth were greater andV:C ratio was smaller in treated piglets than controls. Villus height and crypt depthare indirect indications of the maturity and functional capacity of enterocytes, andmore long the villi and crypts are, a greater number of enterocytes are there present(Hampson, 1986). Baum et al. (2002) also found that villus length was greater inthe small intestine of piglets fed yeast than controls. In poultry fed yeast, Bradleyet al. (1994) found that villus height did not differ from controls, while Zaouche etal. (2000) found differences in the intestinal mucosa in rats given S. boulardii orally.These contrasting results suggest species-specific differences in response to S. boulardiisupplementation.

Considering the multiple functions of the glycoconjugates that make up intestinalmucins, and their differences in health and disease, at least in humans (Rhodes, 1989),we have assessed the mucin content of goblet cells and thickness of the adherent mucousgel. Acid glycoconjugates seemed more abundant in the mucous cells of supplementedthan control piglets. In both treated and control animals sulphated acid glycoconjugateswere abundant in mucous cells of the villi, whereas sialylated glycoconjugates wereabundant in mucus cells of crypts. Sulphated glycoconjugates confer a high viscosity tointestinal mucins, which in such a dense status are potentially able to trap pathogenicbacteria, and so the localization of sulphated glycoconjugates at the surface of the intesti-nal mucosa is potentially very useful in the view of ensuring a local defensive response.Acid glycoconjugates, taken as a whole, help the intestinal mucosa to counteract microor-ganisms and resist to bacterial enzymes (Deplancke and Gaskins, 2001; Montagne etal., 2004). Increased quantities of acid glycoconjugates in yeast-supplemented pigletsmay confer a greater resistance to bacterial infection in the gut in comparison withcontrols.

The histological and histochemical approaches to the quantitative analysis of the intesti-nal adherent mucous gel in pathological as well as experimental conditions is now acceptedas correct, and in some instances preferred to the biochemical one (Sakamoto et al., 2000).Mucous layer is both a barrier and an interface between the lumen and the intestinal epithe-lium; it is also a lubricant and stabilizer of the intestinal microclimate as well as a sourceof energy for the resident microflora (Deplancke and Gaskins, 2001). In healthy animals,the adherent mucous gel produced by goblet cells prevents gut pathogens from invadingthe mucosa (Neutra and Forstner, 1987). In turn, pathogenic microbes secrete glycosi-dases and peptidases to degrade the mucus and gain access to the epithelial cells. Gobletcells usually step up glycoconjugate production in response to the presence of potentialpathogens (Hoskins et al., 1985; Carlstedt-Duke et al., 1986). Mucous secretion is stim-ulated in laboratory mammals by bacterial infection (Cohen et al., 1983; Mantle et al.,1989), bacterial toxins (Roomi et al., 1984), and parasite infestation (Miller et al., 1981).In the present study, the intestinal mucous layer was thicker in control piglets than in sup-plemented animals (P<0.01). This may indicate a greater presence of potential pathogensin the gut lumen of non-supplemented piglets, suggesting that yeast supplementation isable to reduce levels of potential pathogens in the gut. Among the possible mechanismsof this effect, the potential production of microbial growth inhibitors by the yeast is to bementioned. Talarico et al. (1988) showed that the probiotic microorganism Lactobacillusreuteri synthesizes a microbial growth inhibitor. Tang et al. (1999) also suggested a relation


between a thick mucous gel and an increased presence of potential pathogens in pigletintestine.

The thicker mucus layer detected in control animals may also limit the diffusion ofnutrients to the apical surface of epithelial cells, thereby reducing absorption. Thus, thethinner mucus layer in yeast-treated piglets may have contributed to the better feed efficiencyfound in them in comparison with control animals, as also suggested by Patience et al.(1997).

Proliferating cell counts were higher in the mucosa of supplemented piglets than controls.Enterocytes are constantly replaced by division of epithelial cells in crypts. The rate ofreplacement usually matches the rate of loss. Epithelial cells near villus tips are the mostmature and have the greatest digestive and absorptive capacities (Aitken, 1984). In ratsfed a probiotic preparation of Lactobacillus casei and Clostridium butyricum, Ichikawa etal. (1999) reported findings related to proliferating cells similar to those presented here,whereas Baum et al. (2002) found no difference between control weaned piglets and thosetreated with S. boulardii or Bacillus cereus var. Toyoi in terms of number of cells whosenuclei were in S phase.

The greater proliferation rate of intestinal epithelial cells in treated piglets may bedue to the enhanced endoluminal release of polyamines linked to the presence of yeastin the lumen (Buts et al., 1994; Costalos et al., 2003). Polyamines are necessary forcell growth and differentiation, and the demand for them is presumably high in rapidlyproliferating tissues with a high rate of cell turnover such as the intestinal mucosa ofa young animal, instances in which the intracellular concentrations of these substancesincrease (Tabor and Tabor, 1984; Alarcon et al., 1987; Costalos et al., 2003). It is con-ceivable that yeast-supplemented piglets are potentially able to restore the mucosal thin-ning that occurs at weaning (Van Beers-Schreurs et al., 1998) with a better efficiencythan control piglets, as also suggested by Isolauri et al. (2001). This putative effect isnot, however, accompanied by negative consequences on the intestine, since histologi-cal features suggesting pathological changes were never observed. Similarly, Kollman etal. (2001) found no evidence of intestinal hyperplasia in rats after administration of S.boulardii.

Mucosal macrophages were more numerous in supplemented than control piglets. Thissuggests that, in the absence of any evidence of pathological changes in the gut, componentsof the innate defensive system may be more active in treated animals. Since the early phasesof viral infections are mainly counteracted by macrophages (Kosugi et al., 2002), it isconceivable that supplemented piglets are protected against viral infections at a strongerextent than controls.

In conclusion, S. boulardii supplementation is widely used in humans and food animals.The results of the experiment have shown that dietary supplementation is able to modifymorpho-functional aspects of the ileum mucosa in piglets without producing pathologicalor other detrimental changes, and the observed modifications may suggest a way of action ofthis probiotic. These modifications may enable the animals to rapidly overcome some of thenegative consequences of weaning, particularly the largely described mucosal thinning andincreased susceptibility to gastrointestinal disorders. In addition, dietary supplementationwith live S. boulardii seems to be useful in promoting intestinal health, and improving pigletgrowth.


Acknowledgements

Authors wish to thank Dr. Eric Chevaux (Lallemand Sas, France) for cooperation andproviding yeast. Authors are also grateful to Giuseppe Cerri (Azienda Agricola Cavagnone,Buronzo, Italy) for providing facilities for the experiment.

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live yeast dietary supplementation acts upon intestinal morpho

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